The present invention is broadly directed to therapeutic molecules capable of inter alia modulating apoptosis in mammalian cells. The therapeutic molecules of the present invention encompass genetic sequences and chemical entities capable of regulating expression of a novel mammalian gene belonging to the bcl-2 family and which promotes cell survival. The therapeutic molecules of the present invention may have further utility in delaying cell cycle entry. In addition, the present invention extends to chemical entities capable of modulating activity and function of the translation product of said novel gene of the bcl-2 family. The present invention also extends to the translation product of the novel gene of the bcl-2 family and its use in, for example, therapy, diagnosis, antibody generation and as a screening tool for therapeutic molecules capable of modulating physiological cell death or survival and/or modulating cell cycle entry.
Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined following the Bibliography. A summary of the SEQ ID NOs. is provided before the Examples
Throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated dement or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
The increasing sophistication of recombinant DNA technology is greatly facilitating research and development in the medical and allied health fields. This technology is becoming particularly important in research into the treatment and diagnosis of both proliferative cell disorders such as caners and sarcomas and in degenerative diseases such as some autoimmune conditions. There is a need to identify and characterise at the genetic level the elements involved in cell survival and physiological cell death (apoptosis).
Apoptosis is accomplished by a process that is conserved between organisms as diverse as nematodes and man. Positive and negative regulation of cell survival is essential for the proper development and differentiation of the embryo and for ensuring homeostasis in adult tissues. Cell survival can be promoted by the binding of growth factors to their receptors or by interaction of cellular adhesion molecules. A range of cytotoxic agents can counteract these signals and activate apoptosis, a process initially defined by specific morphologic criteria, such as chromatin condensation, cell compaction, membrane blebbing and, often, internucleosomal cleavage of DNA.
The biochemical details of the intracellular pathways governing cell survival and death remain largely undefined. However, several key regulators have emerged. The first to be discovered was Bcl-2, a 26 kD cytoplasmic protein encoded by the bcl-2 gene translocated to the IGH locus in human follicular lymphoma. High levels of Bcl-2 greatly enhance the ability of cells to survive cytokine deprivation and a wide variety of other cytotoxic conditions, including DNA damage.
The mammalian genome contains other genes homologous to bcl-2 but which differ in function. For example, bcl-x blocks apoptosis (Boise et al, 1993) whereas bar and bak inhibit the survival function of bcl-2 and bcl-x (Oltvai et al, 1993; Chittenden et al, 1995; Farrow et al., 1995; Kiefer et al, 1995). Due to the potential importance of cell apoptosis controlling genes in the treatment of cancers and sarcomas and in the treatment of degenerative disorders, there is a need to identify new genes homologous to bcl-2 in structure and function.
In accordance with the present invention, the inventors have identified a novel gene from mammals designated herein xe2x80x9cbcl-wxe2x80x9d. Gene transfer studies show that bcl-w enhances cell survival and belongs to the bcl-2 family of apoptosis-controlling genes. The identification of this new gene will lead to the generation of a range of therapeutic molecules capable of acting as either antagonists or agonists of bcl-w expression or activity and will be useful in cancer or degenerative disease therapy. The identification of the gene will also permit the production of vast quantities of recombinant translation products for use in therapy, diagnosis, antibody generation and as a screen for therapeutic molecules capable of modulating physiological cell deaths or survival including modulating cell cycle entry.
Accordingly, the present invention is directed to a nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding a novel mammalian gene from the bcl-2 family and comprising an amino acid sequence substantially as set forth in SEQ ID NO:7 or SEQ ID NO:9 or having 47% or greater similarity to either of SEQ ID NO:7 or SEQ ID NO:9.
Another aspect of the present invention is directed to a nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:9 or a derivative thereof or encoding an amino acid sequence having 47% or greater similarity to either SEQ ID NO:7 or SEQ ID NO:9.
The term xe2x80x9csimilarityxe2x80x9d as used herein includes em identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, xe2x80x9csimilarityxe2x80x9d includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level xe2x80x9csimilarityxe2x80x9d includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels.
Preferably, the percentage similarity is between 48% and 100% inclusive such as approximately 50% or 55%, 59% or 65%, 70% or 75%, 80% or 85%, 90% or 95% or greater than 96% or a percentage similarity therebetween.
Another aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:6 or SEQ ID NO:8 or a nucleotide sequence encoding an amino acid sequence having 47% or greater similarity to SEQ ID NO:7 or SEQ ID NO:9.
The nucleic acid molecule according to this aspect of the present invention corresponds herein to xe2x80x9cbcl-wxe2x80x9d. This gene has been determined by the inventors in accordance with the present invention to enhance cell survival. The product of the bcl-w gene is referred to as Bcl-w. Human Bcl-w is defined by the amino acid sequence set forth in SEQ ID NO:7 and mouse Bcl-w is defined in SEQ ID NO:9. The respective nucleotide sequences from human bcl-w and mouse bcl-w are shown in SEQ ID NO:6 and SEQ ID NO:8 respectively. Reference herein to xe2x80x9cbcl-wxe2x80x9d includes reference to derivatives thereof includes single or multiple nucleotide substitutions, deletions and/or additions. Similarly, reference herein to xe2x80x9cBcl-wxe2x80x9d includes all derivatives including amino acid substitutions, deletions and/or additions. The gene is preferably from a human, primate, livestock animal (sheep, pig, cow, horse, donkey), laboratory test animal (eg. mouse, at, rabbit, guinea pig), companion animal (eg. dog, cat) or captive wild animal (eg. fox, kangaroo, deer).
Although the present invention relates to a mammalian homologue of Bcl-w having an amino acid sequence of 47% or greater similarity to SEQ ID NO:7 or SEQ ID NO:8, the subject invention does extend to novel Bcl-w homologues from any animal including a mammal previously undisclosed.
Accordingly, another aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding human Bcl-w or a derivative thereof, said human Bcl-w having an amino acid sequence substantially as set forth in SEQ ID NO:7 or is a mammalian homologue thereof having an amino acid sequence of substantially 47% or greater similarity to the amino acid sequence set forth in SEQ D NO:7.
A further aspect provides a nucleic acid molecule comprising a sequence of nucleotides encoding human Bcl-w or a derivative thereof, said murine Bcl-w having an amino acid sequence substantially as set forth in SEQ ID NO:9 or is a mammalian homologue thereof having an amino acid sequence of substantially 47% or greater similarity to the amino acid sequence set forth in SEQ ID NO:9.
The nucleic acid molecule of the present invention is preferably in isolated form or ligated to a vector, such as an expression vector. By xe2x80x9cisolatedxe2x80x9d is meant a nucleic acid molecule having undergone at least one purification step and this is conveniently defined, for example, by a composition comprising at least about 10% subject nucleic acid molecule, preferably at least about 20%, more preferably at least about 30%, still more preferably at least about 40-50%, even still more preferably at least about 60-70%, yet even still more preferably 80-90% or greater of subject nucleic acid molecule relative to other components as determined by molecular weight, encoding activity, nucleotide sequence, base composition or other convenient means. The nucleic acid molecule of the present invention may also be considered, in a preferred embodiment, to be biologically pure.
The nucleic acid molecule encoding bcl-w is preferably a sequence of deoxyribonucleic acids such as cDNA sequence or a genomic sequence. A genomic sequence may also comprise exons and introns. A genomic sequence may also include a promoter region or other regulatory region. In a particularly preferred embodiment, the nucleotide sequence corresponding to bcl-w is a cDNA sequence comprising a sequence of nucleotides as set forth in SEQ ID NO:6 (human) or SEQ ID NO:8 (mouse) or is a derivative thereof including a nucleotide sequence having similar to SEQ ID NO:6 or SEQ ID NO:8 but which encodes an amino acid sequence having 47% or greater similarity to either SEQ ID NO:7 or SEQ ID NO:9.
The term xe2x80x9cderivativexe2x80x9d as used herein includes portions, fragments, parts, homologues or analogues of the nucleic acid molecule or a translation product thereof. A derivative may also be a single or multiple nucleotide or amino acid substitution, deletion and/or addition. A derivative of the nucleic acid molecule of the present invention also includes nucleic acid molecules capable of hybridizing to the nucleotide sequence set forth in SEQ ID NO:6 or SEQ ID NO:8 under low stringency conditions. Preferably, the low stringency is at 42xc2x0 C.
More particularly, the present invention provides a nucleic acid molecule comprising a nucleotide sequence substantially as set forth in SEQ ID NO:6 or SEQ ID NO:8 or a derivative or homologue thereof capable of hybridizing to SEQ ID NO:6 or SEQ ID NO:8 under low stringency conditions and which encodes an amino acid sequence having 47% or greater similarity to the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:9.
Reference herein to a low stringency at 42xc2x0 C. includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1M to at least about 2M salt for hybridisation, and at least about 1M to at least about 2M salt for washing conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01M to at least about 0.15M salt for hybridisation, and at least about 0.01M to at least about 0.15M salt for washing conditions.
The derivatives of the nucleic acid molecule of the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in co-suppression and fusion nucleic acid molecules. Some molecules are also contemplated capable of regulating expression of bcl-w. The present invention also contemplates ribozymes directed to bcl-w. The derivatives of the Bcl-w translation product of the present invention include fragments having particular epitopes or parts of the entire Bcl-w protein fused to peptides, polypeptides or other proteins. Catalytic antibodies are also contemplated to Bcl-w or derivatives thereof. Such catalytic antibodies would be useful for controlling or otherwise modulating Bcl-w. The catalytic antibodies or other regulatory molecules may need to be modified to facilitate entry into the cells. Alternatively, they may be genetically produce in transgenic cells or introduced via a viral or other suitable vector.
In another embodiment the present invention is directed to an isolated nucleic acid molecule encoding bcl-w or a derivative thereof, said nucleic acid molecule selected from the list consisting of:
(I) a nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:9 or having 47% or greater similarity for SEQ ID NO:7 or SEQ ID NO:9;
(ii) a nucleic acid molecule comprising a nucleotide sequence substantially as set forth in SEQ ID NO:6 or SEQ ID NO:8 or comprising a nucleotide sequence encoding an amino acid sequence of 47% or greater similarity to SEQ ID NO:7 or SEQ ID NO:9;
(iii) a nucleic acid molecule capable of hybridizing to the nucleotide sequence substantially set forth in SEQ ID NO:6 or SEQ ID NO:8 under low stringency conditions and encoding an amino acid sequence having of 47% or greater similarity to SEQ ID NO:7 or SEQ ID NO:9;
(iv) a nucleic acid molecule capable of hybridizing to the nucleic acid of part (I) or (ii) or (iii) under low stringency conditions and encoding an amino acid sequence having 47% or greater similarity to SEQ ID NO:7 or SEQ ID NO:9; and
(v) a derivative or mammalian homologue of the nucleic acid molecule of parts (I) or (ii) or (iii) or (iv).
The mammalian homologues contemplated in part (v) of the previous paragraph are novel homologues and do not encompass, for example, known Bcl-2. As stated above, novel homologues of Bcl-w falling outside of the definition herein described are also contemplated y the present invention.
The nucleic acid molecule may be ligated to an expression vector capable of expression in a prokaryotic cell (e.g. E.coli) or a eukaryotic cell (e.g. yeast cells, fungal cells, insect cells, mammalian cells or plant cells). The nucleic acid molecule may be ligated or fused or otherwise associated with a nucleic acid molecule encoding another entity such as a signal peptide, a cytokine or other member of the Bcl-2 family.
The present invention extends to the expression product of the nucleic acid molecule hereinbefore defined.
The expression product is Bcl-w having an amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:9 or is a derivative thereof as defined above or is a mammalian homologue having an amino acid sequence of 47% or greater similarity to the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:9. A derivative may be a single or multiple amino acid substitution, deletion and/or addition. Other derivatives include chemical analogues of Bcl-w. Analogues of Bcl-w contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose confirmational constraints on the proteinaceous molecule or their analogues.
Another aspect of the present invention is directed to an isolated polypeptide selected from the listing consisting of:
(I) a polypeptide having an amino acid sequence substantially as set forth in SEQ ID NO:7 or SEQ ID NO:9 or a sequence having 47% or greater similarity to SEQ ID NO:7 or SEQ ID NO:9;
(ii) a polypeptide encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:6 or SEQ ID NO:8 or a sequence encoding an amino acid sequence having 47% or greater similarity to SEQ ID NO:7 or SEQ ID NO:9;
(iii) a polypeptide encoded by a nucleic acid molecule capable of hybridizing to the nucleotide sequence set forth in SEQ ID NO:6 or SEQ ID NO:8 under low stringency conditions and which encodes an amino acid sequence substantially as set forth in SEQ ID NO:7 or SEQ ID NO:9 or an amino acid sequence having 47% or greater similarity to SEQ ID NO:7 or SEQ ID NO:9;
(iv) a polypeptide as defined in part (I) or (ii) or (iii) in homodimeric form; and
(v) a polypeptide as defied in part (I) or (ii) or (iii) in heterodimeric form.
A derivative may carry a mutation anywhere in the Bcl-w molecule such as but not limited to the S1 and/or S2 region. For example, a substitution at position 94 in S2 from Gly to Glu is encompassed by the present invention. Other areas of Bcl-w for which mutations are contemplated include but are not limited to the region immediately N-terminal to S2, the NH1 region, the S3 region, the S2-S3 region and the BH3 region.
Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.
The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 1.
Crosslinkers can be used, for example, to stabilise 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of Cxcex1 and Nxcex1-methylamino acids, introduction of double bonds between Cxcex1 and Cxcex2 atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
The identification of bcl-w permits the generation of a range of therapeutic molecules capable of modulating expression of bcl-w or modulating the activity of Bcl-2. Modulators contemplated by the present invention includes agonists and antagonists of bcl-w expression. Antagonists of bcl-w expression include antisense molecules, ribozymes and co-suppression molecules. Agonists include molecules which increase promoter ability or interfere with negative regulatory mechanisms. Agonists of Bcl-w include molecules which overcome any negative regulatory mechanism. Antagonists of Bcl-w include antibodies and inhibitor peptide fragments.
The Bcl-w of the present form may be in multimeric form meaning that two or more molecules are associated together. Where the same Bcl-w molecules are associated together, the complex is a homomultimer. An example of a homomultimer is a homodimer. Where at least one Bcl-w is associated with at least one non-Bcl-w molecule, then the complex is a heteromultimer such as a heterodimer. A heteromultimer may include a molecule another member of the Bcl-2 family or a molecule capable of promoting cell survival.
The present invention contemplates, therefore, a method for modulating expression of bcl-w in a mammal said method comprising contacting the bcl-w gene with an effective amount of a modulator of bcl-w expression for a time and under conditions sufficient to up-regulate or down-regulate or otherwise modulate expression of bcl-w. For example, a nucleic acid molecule encoding Bcl-w or a derivative thereof may be introduced into a cell to enhance the ability of that cell to survive, conversely, bcl-w antisense sequences such as oligonucleotides may be introduced to decrease the survival capacity of any cell expressing the endogenous bcl-w gene.
Another aspect of the present invention contemplates a method of modulating activity of Bcl-w in a mammal, said method comprising admit g to said mammal a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease Bcl-w activity. The molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of Bcl-w or its receptor.
Increased bcl-w expression or Bcl-w activity may be influential in regulating inhibition or prevention of cell degeneracy such as under cytotoxic conditions during, for example, xcex3-irradiation and chemotherapy. Decreased bcl-w expression or Bcl-w activity may be important, for example, in selective cancer therapy and increased bcl-w expression may be important for treatment or prophylaxis of conditions such as stroke and Alzheimer""s disease.
Accordingly, the present invention contemplates a pharmaceutical composition comprising a modulator of bcl-w expression or Bcl-w activity and one or more pharmaceutically acceptable carriers and/or diluents.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved at the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as licithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a stile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
When bcl-w and Bcl-w modulators are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ug and 2000 mg of active compound.
The tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.
Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 xcexcg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 xcexcg to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating bcl-w expression or Bcl-w activity. The vector may, for example, be a viral vector.
Conditions requiring modulation of physiological cell death include enhancing survival of cells in patients with neurodegenerative diseases, myocardial infarction, muscular degenerative disease, hypoxia, ischaemia, HIV infection or for prolonging the survival of cells being transplanted for treatment of disease. Alternatively, the antisense sequence could be used, for example, to reduce the survival capacity of tumour cells or autoreactive lymphocytes. The sense sequence may also be used for modifying in vitro behaviour of cells, for example, as part of a protocol to develop novel lines from cell types having unidentified growth factor requirements; for facilitating isolation of hybridoma cells producing monoclonal antibodies, as described below; and for enhancing survival of cells from primary explants while they are being genetically modified.
Still another aspect of the present invention is directed to antibodies to Bcl-w and its derivatives including catalytic antibodies. Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to Bcl-w or may be specifically raised to Bcl-w or derivatives thereof. In the case of the latter, Bcl-w or its derivatives may first need to be associated with a carrier molecule. The antibodies and/or recombinant Bcl-w or its derivatives of the present invention are particularly useful as therapeutic or diagnostic agents. Alternatively, fragments of antibodies may be used such as Fab fragments. Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A xe2x80x9csynthetic antibodyxe2x80x9d is considered herein to include fragments and hybrids of antibodies. The antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be used as a diagnostic tool for assessing apoptosis or monitoring the program of a therapeutic regima.
For example, Bcl-w and its derivatives can be used to screen for naturally occurring antibodies to Bcl-w. These may occur, for example in some autoimmune diseases.
For example, specific antibodies can be used to screen for Bcl-w proteins. The latter would be important, for example, as a means for screening for levels of Bcl-w in a cell extract or other biological fluid or purifying Bcl-w made by recombinant means from culture supernatant fluid. Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays, ELISA and flow cytometry.
It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of Bcl-w.
Both polyclonal and monoclonal antibodies are obtainable by immunization with the protein or peptide derivatives and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of Bcl-w, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See, for example Douillard and Hoffman, Basic Facts about Hybridomas, in Compendium of Immunology Vol II, ed. by Schwartz, 1981; Kohler and Milstein, Nature 256: 495-499, 1975; European Journal of Immunology 6: 511-519, 1976).
Another aspect of the present invention contemplates a method for detecting Bcl-w in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for Bcl-w or its derivatives or homologues for a time and under conditions sufficient for an antibody-Bcl-w complex to form, and then detecting said complex.
The presence of Bcl-w may be accomplished in a number of ways such as by Western blotting, ELISA or flow cytometry procedures. A wide range of immunoassay techniques are available as can be seen by reference to U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These, of course, includes both single-site and two-site or xe2x80x9csandwichxe2x80x9d assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.
Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention the sample is one which might contain Bcl-w including cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid. The sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture.
In the typical forward sandwich assay, a first antibody having specificity for the Bcl-w or antigenic parts thereof, is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducing an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minute) and under suitable conditions (e.g. 25xc2x0 C.) to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.
An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal a bound target may be detectable by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
By xe2x80x9creporter moleculexe2x80x9d as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample. xe2x80x9cReporter moleculexe2x80x9d also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
Alternately, fluorescent compounds, such as fluorecein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope. As in the EIA, the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
The present invention also contemplates genetic assays such as involving PCR analysis to detect bcl-w or its derivatives.
The present invention is further described by reference to the following non-limiting figures and examples.
In the Figures:
FIG. 1 is a representation showing predicted amino acid sequences encoded by murine bcl-w cDNAs (top line, xe2x80x9cBcl-wxe2x80x9d, SEQ ID NO: 9) and chimaeric cDNAs corresponding to transcripts spliced from exon 3 of the bcl-w gene to an exon of the adjacent rox gene (bottom line, xe2x80x9cBcl-w-Roxxe2x80x9d, SEQ ID NO: 10). Boxes highlight the regions of highest homology within the Bcl-2 family, denoted S1, S2 and S3 (Cory, 1995). The arrowhead marks the position corresponding to an intron within the gene. Two residues that differ in human Bcl-w are indicated above the mouse sequence. Not all of the rox cDNA sequences was determined in both orientations.
FIG. 2 is a diagrammatic representation showing the structure of the genomic bcl-w locus and derivation of the bcl-w and bcl-w/rox cDNAs. Overlapping genomic fragments encompassing a 22 kb region were cloned, only one of which (a) is shown. Fragments b to f are subclones of fragment a. Exons are denoted as boxes, with non-coding regions open, the coding region of the bcl-w gene filled and that of the rox gene (see text) stippled. Two types of 5xe2x80x2-end were found for each class of mRNA, suggestive of alternative promoters and/or splicing The first 815 residues of the 3xe2x80x2 untranslated region of bcl-w correspond precisely to those in genomic exon 4; the region not yet sequenced is indicated as a broken line. Restriction mapping suggests the 3xe2x80x2 untranslated region of bcl-w contains at least one more intron. The location of the remainder of the rox gene is not known.
FIG. 3 is a photographic representation showing expression of bcl-w RNA in haemopoietic cell lines. Polyadenylated RNA prepared from the indicated macrophage (mxcfx86), myeloid, and T and B lymphoid lines was fractionated by electrophoresis, transferred to nitrocellulose filters and hybridised with a bcl-w cDNA probe. Probes from the coding region and the bcl-w 3xe2x80x2 untranslated region gave identical results.
FIG. 4 shows the expression of Bcl-w protein. (A) Expression of FLAG-Bcl-w within a clone (D3B5) of FDC-P1 cells transfected with the FLAG-bcl-w PGKpuro expression vector. Transfectants (filled) and parental cells (open) were stained with anti-FLAG monoclonal antibody and analysed by flow cytometry. (B) Immunoblots revealing epitope-tagged survival proteins. Lysates of FDC-P1 cells and FDC-P1 cells expressing FLAG-tagged mouse Bcl-w (clone D3B5), human Bcl-xL or human Bcl-2 were passed over an anti-FLAG affinity gel (Kodak), eluted with FLAG peptide, fractionated by electrophoresis and then analysed with anti-FLAG antibody. (C) Immunoblots with polyclonal rabbit anti-Bcl-w antiserum on cell lysates fractionated by SDS-polyacrylamide gel electrophoresis. In (B) and (C), the stained proteins were visualise by enhanced chemiluminescence (Amersham). WEHI-112.1 and EL4.1 are T lymphoma lines (Harris et al., 1973) and J774 is a macrophage line (Ralph et al., 1975). An additional protein of xcx9c18 kD was also detected by the antiserum, apparently by fortuitous cross-reaction. The molecular weights of markers (Bio-Rad) are given in kD.
FIG. 5 is a graphical representation showing that Bcl-w inhibits apoptosis induced by several but not all cytotoxic agents. FDC-P1 cells, which require IL-3 for survival and proliferation (Dexter et al., 1980), were either (A, left panel) washed three times in medium lacking IL-3 or (A, right panel) irradiated (10 Gy) and then cultured in medium lacking (A, left panel) or containing IL-3 (A, right panel). B6.2.16BW2 T hybridoma cells (Teh et al., 1989) were either cultured in medium containing 1 xcexcM dexamethasone (B, left panel) or irradiated (10 Gy) (B, right panel). CH1 B lymphoma cells (Lynes et al., 1978) were either cultured in the presence of 0.1 xcexcg/ml Jo2 anti-mouse CD95 antibody (Ogasawara et al., 1993) (C, left panel) or irradiated (10 Gy) (C, right panel). Cultures were initiated at 2.5xc3x97105 cells/ml and viability determined by staining with 0.4% w/v eosin on the indicated days.
FIG. 6 is a diagrammatic representation showing that Bcl-w maps in the central region of mouse chromosome 14. The segregation patterns of bcl-w and flanking genes in 134 backcross animals typed for all loci are shown at the top. Each column represents the haplotype inherited from the (C57BL/6Jxc3x97M. spretus) F1 parent; shaded boxes represent the C57BL/6J allele and open boxes the M. spretus allele. The number of offspring inheriting each type of chromosome is listed below each column. A partial chromosome 14 linkage map showing the location of bcl-w in relation to linked genes is shown at the bottom. Recombination distances between loci in centiMorgans are shown to the left of the chromosome and the positions of loci in human chromosomes, where known, are shown to the right References for the human map positions of loci cited in this study can be obtained from GDB (Genome Data Base), a database of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, Md.).
FIG. 7 is a photographic representation showing localisation of bcl-w on human chromosome 14. Partial metaphase showing FISH with the bcl-w intronic probe. (A) Normal male chromosomes stained with propidium iodide. Hybridisation sites on chromosome 14 are indicated by an arrow. (B) the same metaphase as in (A) stained with DAPI for chromosome identification.
FIG. 8 is a representation of a comparison of survival and anti-survival Bcl-2 sub-families. Human Bcl-2 (SEQ ID NO: 11), Bcl-xL (SEQ ID NO: 12), Bcl-w (SEQ ID NO: 7), Bax (SEQ ID NO: 13) and Bak (SEQ ID NO: 14) amino acid sequences were aligned by the Wisconsin PILEUP program. The most conserved portion of the Ced 9 sequence (SEQ ID NO: 15) and a short conserved segment in Bik are also shown. Gaps made in individual sequences to optimise alignment are indicated by dots. Residues identical or very similar (Lxcx9cM; Excx9cD; Kxcx9cR; Vxcx9cI) in the survival-promoting proteins Bcl-2, Bcl-xL and Bcl-w are shown on a black background, as are also those identical or very similar in all the Bcl-2 homologues. A grey background indicates residues shared by Bak and Bax but not present in the survival proteins. Homology regions S1, S2 and S3 (Cory, 1995) and the hydrophobic C-terminal segment are boxed, while the BH1, BH2, BH3 and NH1 regions defined by others (Yin et al., 1994; Subramanian et al., 1995) are overlined. Filled arrowheads indicate conserved residues specific to the survival proteins; open arrowheads, those specific to anti-survival proteins. An unbroken arrow indicates the position of the splice site common to all the proteins; a broken arrow, the position of the alternative 5xe2x80x2 splice that creates the smaller Bcl-x protein and a wavy line a conserved C-terminal motif.
FIG. 9 is a representation of the coding region of (A) human (SEQ ID NO: 6) and (B) murine (SEQ ID NO: 8) bcl-w.